Bcl-2-family proteins play a key role in the control of apoptosis. In particular, gene targeting studies in mice have shown that Bax and Bak (and by analogy, perhaps Bok) are critical effectors;in the absence of these proteins, cells show deficiencies in many forms of apoptosis. Our focus is on how these Bcl-2-family proteins regulate mitochondrial outer membrane permeabilization (MOMP), a primary event in many cell death pathways. MOMP leads to the translocation of cytochrome c and other apoptotic trigger proteins from the mitochondria! inner membrane space into the cytoplasm;these proteins in turn regulate caspase activation and the execution phase of apoptosis. However, even if caspases are inactive or absent, MOMP nevertheless appears to doom most cells to die, through initiating a loss of key mitochondrial functions as well as the generation of reactive oxygen species. Thus, the regulation and mechanism of this process are of critical importance. Here we propose studies that will help elucidate the roles of Bcl-2-family proteins in MOMP. We will use both cell-free systems, to tease apart the mechanisms of action of these proteins, and whole-cell and in vivo approaches, which will extend these investigations to a more physiological context. There are three principal subgroups of the Bcl-2 family: "BH1-4" proteins, which are anti-apoptotic;"BH1-3" proteins, which include the pro-apoptotic family members Bax, Bak and Bok, and the "BH3-only" proteins, which are also pro-apoptotic. The BH3-only proteins are more numerous, are activated specifically through transcriptional and post-translational mechanisms in the context of different cellular stresses, and appear to regulate the other two subfamilies. Our aims, which address each category of the Bcl-2 family in turn, are first, to explore the mechanisms through which the BH3-only proteins regulate the activation of Bax-type proteins;second, to investigate the mechanism of membrane permeabilization by Bax-type proteins;and third, to understand how Bcl-xL, a member of the BH1-4 category, can both prevent MOMP and also reseal the MOM after MOMP has occurred.